Membrane proteins must balance the sequence constraints associated with folding and function against the hydrophobicity required for solvation within the bilayer. We recently found the expression and maturation of rhodopsin are limited by the hydrophobicity of its seventh transmembrane domain (TM7), which contains polar residues that are essential for function. On the basis of these observations, we hypothesized that rhodopsin’s expression should be less tolerant of mutations in TM7 relative to those within hydrophobic TM domains. To test this hypothesis, we used deep mutational scanning to compare the effects of 808 missense mutations on the plasma membrane expression of rhodopsin in HEK293T cells. Our results confirm that a higher proportion of mutations within TM7 (37%) decrease rhodopsin’s plasma membrane expression relative to those within a hydrophobic TM domain (TM2, 25%). These results in conjunction with an evolutionary analysis suggest solvation energetics likely restricts the evolutionary sequence space of polar TM domains.

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Membrane protein variants with diminished conformational stability often exhibit enhanced cellular expression at reduced growth temperatures. The expression of “temperature-sensitive” variants is also typically sensitive to corrector molecules that bind and stabilize the native conformation. There are many examples of temperature-sensitive rhodopsin variants, the misfolding of which is associated with the molecular basis of retinitis pigmentosa. In this work, we employ deep mutational scanning to compare the effects of reduced growth temperature and 9- cis -retinal, an investigational corrector, on the plasma membrane expression of 700 rhodopsin variants in HEK293T cells. We find that the change in expression at reduced growth temperatures correlates with the response to 9- cis -retinal among variants bearing mutations within a hydrophobic transmembrane domain (TM2). The most sensitive variants appear to disrupt a native helical kink within this transmembrane domain. By comparison, mutants that alter the structure of a polar transmembrane domain (TM7) exhibit weaker responses to temperature and retinal that are poorly correlated. Statistical analyses suggest that this observed insensitivity cannot be attributed to a single variable, but likely arises from the composite effects of mutations on the energetics of membrane integration, the stability of the native conformation, and the integrity of the retinal-binding pocket. Finally, we show that the characteristics of purified temperature- and retinal-sensitive variants suggest that the proteostatic effects of retinal may be manifested during translation and cotranslational folding. Together, our findings highlight several biophysical constraints that appear to influence the sensitivity of genetic variants to temperature and small-molecule correctors.

More than 80 loss-of-function (LOF) mutations in the SLC6A8 creatine transporter (hCRT1) are responsible for cerebral creatine deficiency syndrome (CCDS), which gives rise to a spectrum of neurological defects, including intellectual disability, epilepsy, and autism spectrum disorder. To gain insight into the nature of the molecular defects caused by these mutations, we quantitatively profiled the cellular processing, trafficking, expression, and function of eight pathogenic CCDS variants in relation to the wild type (WT) and one neutral isoform. All eight CCDS variants exhibit measurable proteostatic deficiencies that likely contribute to the observed LOF. However, the magnitudes of their specific effects on the expression and trafficking of hCRT1 vary considerably, and we find that the LOF associated with two of these variants primarily arises from the disruption of the substrate-binding pocket. In conjunction with an analysis of structural models of the transporter, we use these data to suggest mechanistic classifications for these variants. To evaluate potential avenues for therapeutic intervention, we assessed the sensitivity of these variants to temperature and measured their response to the proteostasis regulator 4-phenylbutyrate (4-PBA). Only one of the tested variants (G132V) is sensitive to temperature, though its response to 4-PBA is negligible. Nevertheless, 4-PBA significantly enhances the activity of WT hCRT1 in HEK293T cells, which suggests it may be worth evaluating as a therapeutic for female intellectual disability patients carrying a single CCDS mutation. Together, these findings reveal that pathogenic SLC6A8 mutations cause a spectrum of molecular defects that should be taken into consideration in future efforts to develop CCDS therapeutics.